This invention relates in general to molecular biology, pharmacology and medicine and in particular to the isolation, purification and cloning of a human elastase inhibitor.
Preservation of the integrity of local organ function requires a delicate balance of the activities of phagocytic cell proteinases and the action of proteinase inhibitors. Loss of this balance is believed to be a major causative factor in the pathogenesis of asthma, chronic bronchitis, cystic fibrosis, emphysema, sarcoidosis, respiratory distress syndromes, arthritis, certain skin diseases and possibly malignancies. For example, excess release of elastase by neutrophils and monocytes, as well as excess accumulation of monocytes and neutrophils, are believed to be responsible for tissue injury in inflammatory conditions (e.g., arthritis, emphysema) and in neutrophil mediated injury to endothelial cells. The ability to monitor and control the proteinase-proteinase inhibitor balance requires that the relevant proteins be identified, isolated and purified,
An important phagocytic cell proteinase is the serine active site proteinase that is commonly referred to as "neutrophil elastase". Human neutrophil elastase is a glycosylated, 218 amino acid protein that is particularly abundant in neutrophils (0.5% of total protein) and is also found in monocytes and macrophages. The elastase cleaves extracellular matrix proteins, including elastin, proteoglycans, fibronectin, type III and type IV collagen, and certain soluble proteins. The enzyme also is required for neutrophil migration through cell barriers in vitro. The existence of elastase inhibitors in vivo is evident from the neutrophil turnover rate. Despite the fact that neutrophils enter most body sites, a turnover of about 10.sup.11 neutrophils (with a content of about 50 mg elastase) occurs daily in humans without evidence of uncontrolled tissue degradation.
.alpha.1-antitryspin (.alpha.1-AT) is a fast-acting elastase inhibitor. Individuals with genetically reduced levels of .alpha.1-AT (homozygous Z-variant) are predisposed to develop pulmonary emphysema due to uncontrolled elastase action in the third or fourth decade of life. Human .alpha.1-AT currently is used to treat congenital .alpha.1-AT deficiency.
In addition to .alpha.1-AT, other naturally-occurring inhibitors of neutrophil elastase activity have been detected in monocytes and neutrophils in several species. An endogenous elastase inhibitor has been reported in the cytosolic fraction of human blood leukocytes and human lung macrophages (Janoff, A. and Blondin, J., Proc. Soc. Exp. Biol. Med. 136:1050-1053 (1971); Blondin, J. et al., Am. Rev. Resp. Dis. 106:477-479 (1972)). Cytosolic proteins that inhibit elastase have been identified and purified from horse blood neutrophils (Dubin, A., Eur. J. Biochem, 73:429-435 (1977); Potempa, J. et al., J. Biol. Chem, 263:7364 (1988)), pig blood leukocytes (Kopitar, M. and M. Bozic, Acta Pharm. Jugosl. 35:203-212 (1985)) and bovine lung macrophages (Valentine, R. et al., Proc. Soc. Exp. Biol. Med. 168:238-244 (1981)). An elastase inhibitor in the extracellular fluid of cultured guinea pig macrophages has been identified based upon its ability to form a covalent complex with elastase (Remold-O'Donnell, E. and K. Lewandrowski, J. Biol. Chem. 258:3251-3257 (1983)). Larger quantities of the guinea pig elastase inhibitor have been found in macrophage lysates (Remold-O'Donnell, E. and K. Lewandrowski, J. Biol. Chem. 258:3251-3257 (1983)). More recently, a prevalent, fast-acting endogenous elastase inhibitor protein has been detected in mature human monocytes and monocyte-like cells (Remold-O'Donnell, E., J. Exp. Med. 162:2142-2155 (1985)). However, isolation, purification, characterization and cloning of the human monocyte elastase inhibitor has not been reported.